用于生产rHuEPO的无血清培养基的研究

Study on a serum free medium used for production of rHuEPO

观察和筛选了适合工程细胞（Ｃ２细胞）生产重组人促红细胞生成素（ｒｅｃｏｍｂｉｎａｎｔｈｕｍａｎｅｒｙｔｈｒｏｐｏｉｅｔｉｎ，ｒＨｕＥＰＯ）的无血清生产培养基（ＳＦＭ－ｐ）的添加成分。在ＤＭＥＭ∶Ｆ１２（１∶１）培养基中添加了Ｓｅ、乙醇胺、多种维生素、蛋白胨、胰岛素、转铁蛋白和一些细胞因子，构成了这种ＳＦＭ－ｐ。ＳＦＭ－ｐ不含牛血清白蛋白而能维持细胞生长和ｒＨｕＥＰＯ生产。在滚动瓶使用ＳＦＭ－ｐ，细胞的生长和ｒＨｕＥＰＯ的生产都与使用１％ＦＢＳ的培养基无明显区别；在填充床反应器上，用５％ＦＢＳ培养基培养Ｃ２细胞９ｄ后改换ＳＦＭ－ｐ，ＳＦＭ－ｐ能维持Ｃ２细胞在稳定生产状态下达２０ｄ。ｒＨｕＥＰＯ的产率达７１．０ｍｇ／（Ｌ·ｄ）。培养上清的ｒＨｕＥＰＯ最高含量达２８．４μｇ／ｍｌ，葡萄糖耗量达２１ｇ／（Ｌ·ｄ），细胞密度超过３．０×１０７细胞／ｍｌ。

Based on the consumption of medium compositions of CHO cells (C 2) producing the recombinant human erythropoietin (rHuEPO), various additives of serum free medium suitable to C 2 cells were screened. Se, Ethanolamine, lipid, various vitamins, peptone, insulin, transferrin and some cytokines were added in DMEM∶F12 (1∶1) medium, constituting the serum free medium named as SFM p which contained no bovine serum albumin but could support the growth and rHuEPO production of C 2 cells. Productivity of rHuEPO with SFM p was the same as that with 1% FBS medium in rolling bottles. The same studies were conducted in a packed bed bioreactor for C 2 cells using SFM p. C 2 Cells were cultured with 5% FBS medium for 9 days, then substituted with SFM p. Cell culture in SFM p could be maintained in a stable condition of rHuEPO production for 20 days in the bioreactor. rHuEPO Productivity in the bioreactor was 71.0 mg/(L·d), and culture supernatant contained 28.4 μg/ml of rHuEPO. Glucose consumption rate was 21g/(L·d). The highest density of cells could exceed 3.0×10 7 cells/ml. Moreover, rHuEPO Could be easily separated from the culture supernatant. The experiments demonstrate that SFM p can maintain the growth and rHuEPO production of the recombinant C 2 cells.